CN107101981A - A kind of method that utilization utilizing total internal reflection fluorescence microscope detects single biological marker - Google Patents
A kind of method that utilization utilizing total internal reflection fluorescence microscope detects single biological marker Download PDFInfo
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Abstract
The invention discloses a kind of method that utilization utilizing total internal reflection fluorescence microscope detects biological marker.This method is connected and antigen-antibody reaction or the reaction of nucleic acid molecules and probe using biotin-labeled pentylamine, the detection molecules for the fluorescence labeling being fixed in the range of carrier surface 100nm are excited by the fluorescence excitation mode of total internal reflection, fluorescence molecule signal is collected by fluorescence microscope, biological marker number to be detected can be obtained.Because the fluorescence molecule being free in solution is not excited, so the fluorescence signal background noise collected, with very high signal to noise ratio, can tell the fluorescence signal of individual molecule than relatively low, so that this method has high detection sensitivity.This detection method can be used for detecting various biological markers, and the preventing and treating of discovery and disease for cause of disease in disease is significant.
Description
Technical field
The present invention relates to technical field of biological, it is inverted in particular to one kind using based on total internal reflection fluorescent
The method that microscope detects single biological marker.
Background technology
The disease that many is triggered by pathogenic microorganism occurs and propagated in the range of the world today extensively, seriously endangers and arrives people
Class and the health of animals and plants, influence the quality of the life of the mankind.The attribute of pathogenic microorganism is understood, for prevention and treatment of diseases
It is essential link.The method of detection cause of disease has much at present, such as EUSA (ELISA), Western blotting
(Western Blot) and PCR (PCR) etc. are tested, but these methods more or less all be present,
Such as complex operation, cost are high, the low shortcoming of sensitivity.Bio-tissue or intracellular micro cause of disease use conventional method
It is difficult to detect by coming, this, which has been resulted in, can not determine infected animal optimal treatment means, or even when can miss optimal treatment
Machine, causes illness to aggravate.Therefore, detect that cause of disease is most important with treating for control and prevention of disease efficient and sensible.
Total internal reflection (total internal reflection) is a kind of optical phenomena.Light is higher from refractive index
The relatively low optically thinner medium of optically denser medium (such as glass, refractive index 1.52) entrance refractive index (such as liquid solution, refractive index 1.33~
1.38), incidence angle is θ 1, and refraction angle is θ 2, and when there is n1sin θ 1=n2sin θ 2, portions incident light is reflected, portion
The raw reflection of distribution.When incidence angle θ 1 is more than or equal to critical angle θ c, θ 1 >=θ c, now light no longer transmit into medium 2, hair
Total reflection is given birth to.
When total internal reflection occurs, a kind of evanescent wave with special nature can be produced, the intensity of this evanescent wave is in sample
Exponentially decay along the Z axis of vertical interface in the aqueous phase of interface, thus be merely able to excite using this fluorescence excitation mode and be fixed on
Fluorescence molecule in the range of the 100nm of cover glass surface, the fluorescence molecule being free in the solution light that will not be excited is excited, so adopting
Collect optical signal background noise than relatively low.The image background obtained using total internal reflection mode of excitation is very low, with sufficiently high
Signal to noise ratio, can tell the fluorescence signal of individual molecule.Therefore, can be to the list of cause of disease using utilizing total internal reflection fluorescence microscope
Individual biological marker is observed, with high detection sensitivity.In the present invention, we disclose a kind of using based on complete interior
The method that the fluorescence inverted microscope of reflection detects biological marker, the method is simple to operate, and cost is low, and with very high
Detection sensitivity, it is significant for Pathogen test and disease treatment.
The content of the invention
It is an object of the invention to provide a kind of method of fixed biological marker.
It is a kind of using based on the single life of total internal reflection fluorescent inverted microscope detection another object of the present invention is to provide
The method of thing marker.This method is connected indirectly using the detection molecules of fluorescence labeling with biological marker, by complete interior anti-
The fluorescence for penetrating the detection molecules of fluorescence microscope collection fluorescence labeling is counted out, and reaches the purpose of detection biological marker.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of fixed biological marker, comprises the following steps：
(1) surface of carrier is first modified with biotinylation PEG；
(2) add between Avidin, biotin and avidin and can connect on described carrier；
(3) again to the attachment 1 that biotin labeling is added on described carrier, connect between biotin and avidin
Connect, described attachment 1 is fixed on described carrier；
(4) attachment 2 is added, is combined with described attachment 1；
(5) biological marker is added, biological marker can be connected with described attachment 2, and described attachment 2 is made
Biological marker is captured for bait；
(6) add the attachment 3 reacted with biological marker, be eventually adding the detection molecules of fluorescence labeling with it is described
Attachment 3 is connected, biological marker is fixed on by specific reaction between biological marker and attachment it is described on carrier,
Form the single biological marker detection pattern of a kind of " sandwich ".
The biological marker is one kind in DNA, RNA, protein, biological micromolecule, cell, bacterium and virus；Institute
Attachment 1 is stated for probe or secondary antibody；Described attachment 2 is probe or monoclonal antibody；Described attachment 3 is probe or resisted more；Institute
It is the probe of fluorescence labeling or the secondary antibody of fluorescence labeling to state the detection molecules of fluorescence labeling.
When described biological marker is DNA, RNA, the attachment 1 is probe, and described attachment 2 is probe, institute
The attachment 3 stated is probe, and the detection molecules of the fluorescence labeling are the probe of fluorescence labeling；Described biological marker is egg
When white matter, cell, bacterium or virus, the attachment 1 is secondary antibody, and described attachment 2 is monoclonal antibody, and described attachment 3 is
Resist, the detection molecules of the fluorescence labeling are the secondary antibody of fluorescence labeling more.
Described Avidin is Streptavidin.
Described biological marker can be albumen, the PCV2cap albumen (i.e. antigen PCV2) of such as pig circular ring virus.Make
For a kind of technical scheme, when described biological marker is the PCV2cap albumen of pig circular ring virus, described attachment 1 is sheep
Anti- mouse secondary antibody, described attachment 2 is mouse source monoclonal antibody, and described attachment 3 resists more for rabbit source, the detection point of the fluorescence labeling
The goat-anti rabbit secondary antibody that son marks for fluorescein isothiocynate (FITC).
Described carrier is transparent carrier, as a kind of optimal technical scheme, and described carrier of quartz slide by being made up
Sample capsule, the Loading channel provided with closing and the well for being loaded to Loading channel in described sample capsule.
The detailed preparation process of above-mentioned sample capsule is：
A, multiple holes are bored as well on slide and are cleaned repeatedly with deionized water；
B, with volume ratio 1:1 acetone/alcoholic solution is rinsed with deionized water again after being cleaned by ultrasonic slide and cover glass
Totally；
C, be cleaned by ultrasonic slide and cover glass with 1mol/L potassium hydroxide solution after use deionized water rinsed clean again
And dry；
D, described slide and cover glass are placed in volume ratio is 125:5:1 methanol/anhydrous acetic acid/amino silane is even
Join and stand reaction in agent mixed liquor after ultrasound；It is subsequently placed in methanol after ultrasound and stands reaction；Deionized water rinsed clean is used again
And dry；
E, take biotin-PEG and mPEG to be dissolved in 0.1mol/L sodium bicarbonate aqueous solution and go bubble removing to obtain PEGylation
Reagent；Amount ratio (the mg of biotin-PEG, mPEG and 0.1mol/L sodium bicarbonate aqueous solution:mg:Ul it is) 1:40:320.
F, by after cover glass frame placing flat, PEGylation reagent droplet is added dropwise in centre position thereon, slide is aligned close
Drop, slide and cover glass can be together with the automatic suctions in the presence of liquid tension；
G, the slide being attached together and cover glass are incubated overnight after in deionization current by slide and Gai Bo
Piece is rinsed well and dried；
H, spacing parallel arranging paste multistage two-sided tape on slide, and multiple channel grooves are vertically divided on slide,
Cover glass is just sticked on two-sided tape to channel groove and adhesive tape is compressed so that mutual not insertion between adjacent channel recesses；With
The two ends that epoxy resin seals the channel groove form the Loading channel of closing, and described hole is respectively provided at institute as well
Stating the two ends of Loading channel is used to be loaded into Loading channel.
Adopt the carrier for being fixed with biological marker prepared with the aforedescribed process.
The above-mentioned carrier for being fixed with biological marker is detecting single biological label using utilizing total internal reflection fluorescence microscope
Application in thing, the fluorescence molecule of the carrier surface is observed using utilizing total internal reflection fluorescence microscope, by gathering fluorescence molecule
Signal accurately detects fixed biological marker number on the carrier.
A kind of method that utilization utilizing total internal reflection fluorescence microscope detects single biological marker, adopting with the aforedescribed process will be raw
Thing marker is fixed on described carrier, and the fluorescence molecule of carrier surface is observed using utilizing total internal reflection fluorescence microscope, is passed through
Accurately detection is fixed on the biological marker number on carrier to collection fluorescence molecule signal.
The method that the present invention detects single biological marker using utilizing total internal reflection fluorescence microscope, by biotinylation attachment
1 is fixed to the coated carrier surface of Streptavidin, and biological marker to be checked is captured indirectly (for example, nucleic acid molecules, antigen PCV2
Deng), biological marker can be connected with the detection molecules of fluorescence labeling indirectly again, pass through the mode excitation fluorescence based on total internal reflection
Group, fluorescence molecule signal is gathered using fluorescence inverted microscope, obtains the detection of fluorescence labeling reacted with biological marker
Molecule amount, biological marker number can be obtained by removing gained fluorescence molecule number after background value, and this biological marker is various
Cause of disease to be checked in disease.
Various reagents needed for detection are added on transparent carrier step by step with biological marker to be checked, based on total internal reflection
Fluorescence inverted microscope can only excite the fluorescent labeled antibody of transparent carrier surface 100nm scopes, gather fluorescence molecule signal
Biological marker number can accurately be detected.The inventive method can effectively exclude the background of more interference, also to suspend
Fluorescent donor molecule in solution will not be excited and send fluorescence.
The method of fixed biological marker of the present invention is that biological marker is fixed into carrier (such as quartzy glass
Piece) on method.Carrier in the present invention is provided with closing in the sample capsule being made up of quartz slide, described sample capsule
Loading channel and well for being loaded to Loading channel.Described sample capsule is by one piece of slide and one piece of lid glass
Piece is made, can by the reaction of linking between Avidin and biotin, and antigen-antibody reaction or nucleic acid molecules and probe
To be fixed to carrier surface after specific biological marker is captured.
The method have the characteristics that 1, detecting step is convenient and easy, it is only necessary to using liquid-transfering gun step by step by reagent and life to be checked
Thing marker is added on carrier, is then observed using utilizing total internal reflection fluorescence microscope；2nd, detection time is short, and inspection is drawn from being loaded onto
Surveying less than 1 hour of concentration can complete；3rd, testing cost is low, detects that the carrier used can be prepared by two pieces of quartz slides,
Detection amount of antibody used is seldom；4th, sensitivity is high, and utilizing total internal reflection fluorescence microscope is observed that single biological marker, needs
Want sample concentration to be checked low.
Beneficial effects of the present invention：
The inventive method connected using biotin-avidin and antigen-antibody reaction or nucleic acid molecules and probe it is anti-
Should, the detection point for the fluorescence labeling being fixed in the range of carrier surface 100nm is excited by the fluorescence excitation mode of total internal reflection
Son, fluorescence molecule signal is collected by fluorescence microscope, can obtain biological marker number to be detected.It is molten due to being free on
Fluorescence molecule in liquid is not excited, so the fluorescence signal background noise collected is than relatively low, with very high signal to noise ratio,
The fluorescence signal of individual molecule can be told, so that this method has high detection sensitivity.This detection method can be used for inspection
Various biological markers are surveyed, the preventing and treating of discovery and disease for cause of disease in disease is significant.
Brief description of the drawings
Fig. 1 sample capsule assembling figures.
Fig. 2 detects single biological marker schematic diagram using utilizing total internal reflection fluorescence microscope：
Wherein, biotinylated antibody is the sheep anti mouse secondary antibody of biotin labeling, and biomarker to be detected is pig annulus
Viral 2 types (PCV2cap) albumen, the antibody of fluorescence labeling is the goat-anti rabbit secondary antibody of fluorescein isothiocynate (FITC) mark.
Fig. 3 is to detect single antigen PCV2 testing results using utilizing total internal reflection fluorescence microscope
Wherein, A is that microscope imaging picture B is result histogram
Fig. 4 is the line chart (figure C) and linear regression analysis result of fitting after antigen PCV2 extension rates are taken the logarithm
(figure D)
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material therefor, reagent in following embodiments, unless otherwise specified, are commercially obtained.
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.
Experiment in following examples, is respectively provided with more than three times and repeats experiment, results averaged.
Embodiment 1 makes the sample capsule for sample-adding
1st, 10 diameter 0.8mm hole is bored in thickness 1mm slide using normal drill bit, this hole is especially suitable for
200ul yellow pipette tips are loaded；
2nd, slide is rinsed well repeatedly with deionized water；
3rd, with volume ratio 1:1 acetone/alcoholic solution is cleaned by ultrasonic slide and cover glass 20min；
4th, rinsed three times with deionized water；
5th, it is cleaned by ultrasonic 20min with 1mol/L potassium hydroxide solution；
6th, rinsed three times with deionized water；
7th, slide and cover glass are dried in an oven；
8th, by slide and cover glass be placed in methanol containing 250ml, 10ml anhydrous acetic acids, 2ml amino silicane coupling agents it is mixed
Liquid ultrasound 1min is closed, reaction 20min is then stood；
9th, slide and cover glass are placed in ultrasound 1min in methanol, then stand reaction 10min；
10th, rinsed three times with deionized water；
11st, slide and cover glass are dried in an oven；
12nd, 1mg biotin-PEG and 40mg mPEG is taken to be dissolved in 320ul 0.1mol/L sodium bicarbonate aqueous solution,
7200g centrifugations 1min removes bubble removing；
13rd, 66ul PEGylation reagent droplets are added dropwise in centre position thereon in cover glass frame placing flat in super-clean bench, will
Slide is aligned close to drop, slide can be in automatic suction in the presence of liquid tension with cover glass together with；
14th, the slide being attached together and cover glass frame are incubated on support, being placed in the metallic aluminium box that water is arranged at bottom
Educate overnight；
15th, slide and cover glass are rinsed well in deionization current, dried in an oven；
16th, 6 sections of two-sided tapes are pasted on slide, 10 holes of slide are vertically divided into 5 passages side by side recessed
Groove；Then cover glass is just sticked on two-sided tape to groove, adhesive tape is compressed with yellow pipette tips so that between adjacency channel mutually not
Insertion；The two ends for sealing channel groove with epoxy resin form the Loading channel of closing so that the two ends point of each Loading channel
Not Dui Ying two circular cavities, described hole is used to be loaded as well.
Cause of disease molecule is fixed the substep sample-adding of the antibody of embodiment 2 and antigen to be detected, and using based on complete interior anti-
The fluorescence inverted microscope penetrated obtains cause of disease molecule amount
1st, take and be dissolved in T50 (10mmol/L Tris-HCl, (pH 8.0) and 50mmol/L NaCl) solution
0.2mg/mL Streptavidins 10ul is added in the Loading channel of sample capsule, is incubated at room temperature 5min；Biotin and avidin it
Between will connect；
2nd, take 20ul T50 solution to add Loading channel, wash away uncombined Streptavidin；
3rd, the 10ul sheep anti mouse secondary antibody of 40nM biotin labelings is added to Loading channel, is incubated at room temperature 15min；Biotin
It will be connected between Avidin, secondary antibody is secured to passage surface；
4th, (the 0.1mg/ml bovine serum albumin(BSA)s (BSA) in T50 solution are dissolved in, BSA can be with 20ul T50-BSA
Prevent albumen non-specific adsorption on slide) add passage cleaning twice, wash away uncombined secondary antibody；
5th, take 10ul 20nM mouse source monoclonal antibody to add Loading channel, be incubated at room temperature 15min；Mouse source monoclonal antibody and two anti-bindings,
Mouse source monoclonal antibody captures antigen PCV2 as bait；
6th, add Loading channel with 20ul T50-BSA to clean twice, wash away uncombined monoclonal antibody；
7th, take 10ul antigen PCV2 to add Loading channel, be incubated at room temperature 15min；Antigen PCV2 will be with capture antibody hair
Raw connection；
8th, add Loading channel with 20ul T50-BSA to clean twice, wash away uncombined albumen
9th, the 10ul how anti-addition Loading channel in 10nM rabbits source is taken, 15min is incubated at room temperature；It is allowed to react with antigen PCV2；
10th, add Loading channel with 20ul T50-BSA clean twice, wash away uncombined more anti-；
11st, goat-anti rabbit secondary antibody (the goat-anti rabbit two of fluorescein isothiocynate (FITC) mark of 10ul 2nM fluorescence labelings is taken
It is anti-) Loading channel is added, it is incubated at room temperature 15min；It is allowed to and the how anti-connection in rabbit source.It is anti-by specificity between antigen PCV2 and antibody
In the Loading channel that antigen PCV2 should be fixed on to the sample capsule, the antigen molecule detection pattern of a kind of " sandwich " is formed；
12nd, add Loading channel with 20ul T50-BSA to clean twice, wash uncombined rabbit secondary antibody off.
13rd, using the excitation FITC of 488nm wavelength, fluorescence point is gathered by total internal reflection fluorescent inverted microscope
Subsignal；
14th, antigen is not added with as parallel control in another sample capsule passage, obtain background value.By complete interior anti-
The fluorescence molecule signal that fluorescence microscopy is arrived is penetrated, background value in control is subtracted, the fluorescence molecule number of final gained is exactly
The biological marker number detected.
The inventive method has high detection sensitivity, and experimental result shows and (is shown in Table 1), with antigen PCV2 extension rates
Increase variance yields constantly reduces, and illustrates that data tend towards stability, the repeated effect of experiment is also more preferable, when antigen PCV2 diluted concentrations
For 10 times when, variance yields is larger, and data are relatively discrete, is due to antigen PCV2 excessive concentration, antigen PCV2 completely and
Mouse source monoclonal antibody is combined, and excessive antigen PCV2 may produce faint non-specific binding with fluorescence antibody, cause to count
As a result there is deviation.But, we are concerned with whether it can also be diluted here, and the phosphor dot meter under the conditions of dilution for many times
Several stability and repeatability.
The testing result of table 1 not under synantigen PCV2 extension rates
| Antigen PCV2 extension rates | Background fluorescence is counted | Phosphor dot mean number | Variance |
| 10 | 149 | 537 | 11.08107 |
| 20 | 152 | 394 | 8.90034 |
| 40 | 147 | 249 | 4.77842 |
| 80 | 150 | 190 | 7.01277 |
| 100 | 152 | 152 | 2.15022 |
| 200 | 148 | 150 | 1.86173 |
Four kinds of antibody are individually added into come the background value of confirmatory experiment passage by the way of substep is loaded, to background signal figure
As carrying out image procossing, the number of its phosphor dot is calculated, average is obtained for 150.It was found that the number of average is between antigen PCV2
After 80 times of dilution, tested by being repeated several times, when diluted concentration is more than 100, when reaching 200 times, the number of phosphor dot
Change is less (as shown in Figure 3, Figure 4).It therefore, it can the limit of estimation detection between 80 times and 100 times of dilution, at 80 times
And taking concentration gradient to be diluted between 100 times, when concentration gradient takes 95 times, the mean number of phosphor dot is 160.
Detectable limit scope, is detection when antigen PCV2 final concentration takes 20nM to be diluted between 90 to 100 times according to a preliminary estimate
Limit range, for the stability of confirmatory experiment, we are to 90 times, 95 times, and 100 times of diluted concentrations have carried out multiplicating property
Experiment, every time experiment takes 20 width images to carry out the counting of phosphor dot, obtains average value.
Antigen PCV2 initial concentration is 2.8 × 10 in embodiment-4Mg/mL, in this, as the starting point of dilution, when dilution 95
Times when, concentration be 2.9 × 10-6mg/mL.Copy number just refers to certain gene (can be plasmid) in a certain biological genome
In number.Single copy is exactly gene only one of which in the biological genome, and at most referring to has multiple.Copy number calculates public
Formula：Copy/ml=6.02 × 1023(Avgadro constant) copy number/mole × (concentration g/mL)/(MW g/mol), we
Known PCV2 DNA molecular amount is 28000g/mol.Be brought into formula and converted, can proper diluted concentration be 2.9 × 10- 6When mg/mL, it is about 6.24 × 10 to be converted into copy unit10Copy/mL.Our unimolecule pulldown method detection limits
It is smaller, represent in the blood sample of equal amount pig, smaller virus quantity can be detected, and this is illustrated under unimolecule
Pull technology is accurate, and therefore, we can be in this approach as the new method of business development, so that in order to detect that pig circular ring virus is carried
For relatively reliable technical support.The problem of unresolved agriculture field, provides strong support.
Claims (10)
1. a kind of method of fixed biological marker, it is characterised in that：Comprise the following steps：
(1) surface of carrier is first modified with biotinylation PEG；
(2) add between Avidin, biotin and avidin and can connect on described carrier；
(3) again to the attachment 1 that biotin labeling is added on described carrier, connected between biotin and avidin, institute
The attachment 1 stated is fixed on described carrier；
(4) attachment 2 is added, is combined with described attachment 1；
(5) biological marker is added, biological marker can be connected with described attachment 2, described attachment 2, which is used as, to lure
Bait captures biological marker；
(6) attachment 3 reacted with biological marker is added, the detection molecules of fluorescence labeling and described connection is eventually adding
Thing 3 is connected, and biological marker is fixed on to described on carrier, formation by specific reaction between biological marker and attachment
A kind of single biological marker detection pattern of " sandwich ".
2. according to the method described in claim 1, it is characterised in that：The biological marker is DNA, RNA, protein, biology
One kind in small molecule, cell, bacterium and virus；The attachment 1 is probe or secondary antibody；Described attachment 2 be probe or
Monoclonal antibody；Described attachment 3 is probe or resisted more；The detection molecules of the fluorescence labeling are the probe or fluorescence mark of fluorescence labeling
The secondary antibody of note.
3. method according to claim 2, it is characterised in that：When described biological marker is DNA, RNA, the connection
Thing 1 is probe, and described attachment 2 is probe, and described attachment 3 is probe, and the detection molecules of the fluorescence labeling are glimmering
The probe of signal；When described biological marker is protein, cell, bacterium or virus, the attachment 1 is secondary antibody, institute
The attachment 2 stated is monoclonal antibody, and described attachment 3 is resists more, and the detection molecules of the fluorescence labeling are the secondary antibody of fluorescence labeling.
4. according to the method described in claim 1, it is characterised in that：Described Avidin is Streptavidin.
5. according to any described method in Claims 1 to 4, it is characterised in that：Described biological marker is pig circular ring virus 2
During the PCV2cap albumen of poison, described attachment 1 is sheep anti mouse secondary antibody, and described attachment 2 is mouse source monoclonal antibody, described company
Connect thing 3 for rabbit source to resist, the detection molecules of the fluorescence labeling are the goat-anti rabbit secondary antibody that fluorescein isothiocynate (FITC) is marked more.
6. according to the method described in claim 1, it is characterised in that：Described carrier is transparent carrier, is used as a kind of preferred skill
Art scheme, described carrier is that the sample-adding provided with closing leads in the sample capsule being made up of quartz slide, described sample capsule
Road and the well for being loaded to Loading channel.
7. method according to claim 6, it is characterised in that：The detailed preparation process of the sample capsule is：
A, multiple holes are bored as well on slide and are cleaned repeatedly with deionized water；
B, with volume ratio 1:1 acetone/alcoholic solution uses deionized water rinsed clean again after being cleaned by ultrasonic slide and cover glass；
C, it is cleaned by ultrasonic with 1mol/L potassium hydroxide solution after slide and cover glass and with deionized water rinsed clean and is dried again
It is dry；
D, described slide and cover glass are placed in volume ratio is 125:5:1 methanol/anhydrous acetic acid/amino silicane coupling agent
Reaction is stood in mixed liquor after ultrasound；It is subsequently placed in methanol after ultrasound and stands reaction；With deionized water rinsed clean and dry again
It is dry；
E, take biotin-PEG and mPEG be dissolved in 0.1mol/L sodium bicarbonate aqueous solution and go bubble removing obtain PEGylation examination
Agent；
F, by after cover glass frame placing flat, PEGylation reagent droplet is added dropwise in centre position thereon, slide is aligned close to liquid
Drop, slide can be in automatic suction in the presence of liquid tension with cover glass together with；
G, will the slide that be attached together and cover glass be incubated overnight after slide and cover glass rushed in deionization current
Wash clean is simultaneously dried；
H, spacing parallel arranging paste multistage two-sided tape on slide, and multiple channel grooves are vertically divided on slide, will be covered
Slide just sticks on two-sided tape to channel groove and compresses adhesive tape so that mutual not insertion between adjacent channel recesses；Use epoxy
The two ends of channel groove described in resin seal form the Loading channel of closing, described hole as well be respectively provided at it is described plus
The two ends of sample passage are used to be loaded into Loading channel.
8. using the carrier for being fixed with biological marker that in claim 1~7 prepared by any described method.
9. the carrier for being fixed with biological marker described in claim 8 is detecting single life using utilizing total internal reflection fluorescence microscope
Application in thing marker, it is characterised in that：The fluorescence molecule of the carrier surface is observed using utilizing total internal reflection fluorescence microscope,
Fixed biological marker number on the carrier is accurately detected by gathering fluorescence molecule signal.
10. a kind of method that utilization utilizing total internal reflection fluorescence microscope detects single biological marker, it is characterised in that：Using right
It is required that biological marker is fixed on described carrier by any described method in 1~7, utilizing total internal reflection fluorescence microscope is utilized
The fluorescence molecule of carrier surface is observed, accurately detection is fixed on the biological marker number on carrier by gathering fluorescence molecule signal
Mesh.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111537480A (en) * | 2020-04-26 | 2020-08-14 | 中央民族大学 | Rapid virus detection method based on single-molecule total internal reflection fluorescence imaging technology |
| CN112415184A (en) * | 2019-08-22 | 2021-02-26 | 香港科技大学 | Single molecule separation system for cell populations and single cells, and methods and uses thereof |
| CN114486830A (en) * | 2022-01-24 | 2022-05-13 | 复旦大学 | System and method for single-molecule protein and biomolecule counting in single cell |
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